摘要
目的体外合成和筛选特异性阻断人滑膜成纤维细胞(RASF)环氧合酶-2(hCOX-2)的小分子干扰RNA(siRNA),同时了解COX-2抑制对炎症因子表达水平的影响。方法设计4条针对人COX-2 mRNA siRNA(1#～4#siRNA),1条随机序列作为对照。分成A～H组,A组为空白阴性对照,B～F组处理依次为随机siRNA、1#～4#siRNA。应用LipofectAMINE 2000将上述siRNA转染入RASF,各培养孔加入100 nmol/L的佛波酯。转染48h后,分别应用反转录聚合酶链式反应(RT-PCR)和Western Blot检测hCOX-2 mRNA和蛋白表达水平。采用酶联免疫吸附(ELISA)方法检测各组上清液中前列腺素E_2(PGE_2)和白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α、血管内皮生长因子(VEGF)水平。结果4#siRNA转染的RASF表达hCOX-2 mRNA和蛋白水平明显低于其他siRNA干预组和阴性对照,且其上清液PGE_2和IL-1β、IL-6、TNF-α、VEGF水平较其他各组明显下降。结论4#siRNA能有效抑制COX-2 mRNA表达和COX-2蛋白的合成,且上清液中PGE_2和IL-1β、IL-6、TNF-α及VEGF水平最低。
Objective To design, synthesize and screen high efficient small interfering RNA （siRNA） targeting to cyclooxygenase-2 （COX-2） on rheumatoid arthritis synovial fibroblasts （RASF）. To further study the effect of specific COX-2 siRNA interfering on mediators of inflammatory cytokines. Methods Four pairs of siRNA for human COX-2 mRNA were synthesized by utilizing RNA design software, while another random sequence was designed as control. They were divided into group A to H. Among them, group A was used as the negative control （CTL）, and group B to F were transfected as random siRNA （NC）, 1#-4#siRNA in order. These siRNAs were transferred into RASF by LipofectAMINE2000 package and PMA （phorbol-12-myristate-13-acetate） was added into each culture and with a final concentration of 100 nmol/l. RASF was collected 48 hours after transfection. The expression of hCOX-2 at mRNA level was determined by reverse transcription-polymerase chain reaction （RT-PCR） and hCOX-2 protein level by Western Blot. The supernatant levels of PGE2, IL-1β IL-6, TNF-α and vascular endothelial growth factor （VEGF） of the above groups were detected by ELISA. Results The levels of hCOX mRNA and protein in RASF treated with 4#siRNA were significantly lower than those of the negative control and other groups. The level of PGE2 and cytokines like IL-1β IL-6, TNF-α and VEGF in the supernatant were lower in the 4#siRNA group than in other groups. Conclusion 4#siRNA can effectively inhibit the expression of COX-2 mRNA and the synthesis of the COX-2 protein in human synovial fibroblasts. The level of PGE2, IL-1β IL-6, TNF-α and VEGF is the lowest in the supernatant. Thus 4#siRNA has been confirmed to specifically block the COX-2 in human synovial fibroblasts.
出处
《中华风湿病学杂志》
CAS
CSCD
2007年第5期275-279,共5页
Chinese Journal of Rheumatology
